Oligonucleotides are used in a multitude of research and clinical applications. Ensuring the integrity of the oligonucleotide is vital to prevent failures or misleading results in applications using the oligonucleotides. Several methods have been developed to ensure that the resulting oligonucleotide is what the technician intended for use in their application.
One method of ensuring quality during the synthesis of the oligonucleotide is through trityl monitoring. The dimethoxytrityl (DMT) group that is used for capping the 5′-hydroxyl group of the monomers in the oligonucleotide synthesis fluoresces in its protonated form after it is removed with an acid. The absorbance of the fluorescence can be measured at or around 498 nm. A decrease in the absorbance level can be an indication that coupling was inefficient.
The identity of an oligonucleotide target can be assessed post-synthetically by measuring the predominant molecular weight of the population. The target sequence is known, so the calculated molecular weight of the bases themselves is the standard by which one compares the measured molecular weight to see if the desired compound was created. One way of utilizing this principle is through matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). MALDI-TOF uses laser light in conjunction with a chemical matrix to impart a charge to the sample in question and repel it from the sample plate. The resulting ions travel through a flight tube to the detector, which measures particle counts as a function of time. The time-of-flight (TOF) is directly proportional to the mass of the molecule.
MALDI-TOF is a robust and incredibly high-throughput process for assessing molecular weight. One drawback of MALDI-TOF is that the ionization efficiency (and therefore the resolution) of the procedure drops rapidly above ˜45 bases or >13,000 Da. With the popularity of 70 mer arrays and long oligonucleotides for cloning and/or gene synthesis, another method is needed to assess longer products.
Electrospray ionization (ESI) mass spectroscopy ionizes target molecules such as oligonucleotides into multiple charge states. The readout of these charge states is a waveform that can be deconvoluted into parent peaks. The method uses a tight m/z window of 500-1,500, which gives it high mass accuracy. As only the charge state will vary for the ions, oligonucleotides with high molecular weights can be analyzed using this method. Therefore ESI is often a preferred quality control (QC) method over MALDI-TOF for longer oligonucleotides (see Elliott, B. and Hail, M. High-Throughput Analysis of Oligonucleotides Using Automated Electrospray Ionization Mass Spectrometry American Biotechnology Laboratory, January 2004).
Currently the use of ESI and MALDI-TOF in quality control is limited to comparing the results of the ESI to the expected peak that would result from an oligonucleotide sequence of that given molecular weight. Some assays can use 48, 96 or greater oligonucleotide sequences for the given assay, and a given sequence may be indistinguishable from other sequences in the assay based upon molecular weight alone. The quality control of an assay is therefore reliant on running the assay through the given platform to ensure that the results of the assay equate to the expected results.
Other methods are known in the art that observed multi-oligonucleotide samples to detect the presence or absence of a single nucleotide polymorphism (see Koster et al., U.S. Pat. No. 7,074,563), but this assay can not detect the presence or absence of any constituent oligonucleotide in an oligonucleotide mixture and its relative concentration therein.
The proposed method involves generating a theoretical MALDI-TOF or ESI trace (fingerprint) of a multi-oligonucleotide sample and then comparing the actual MALDI-TOF or ESI data of the mix to the fingerprint as a QC check.